This invention relates to novel nucleoside or nucleotide analogs, processes for their synthesis and incorporation into polynucleotides.
The following is a brief description of nucleoside analogs. This summary is not meant to be complete but is provided only for an understanding of the invention that follows. This summary is not an admission that all of the work described below is prior art to the claimed invention.
Nucleoside modifications of bases and sugars, have been discovered in a variety of naturally occurring RNA (e.g., tRNA, mRNA, rRNA; reviewed by Hall, 1971 The Modified Nucleosides in Nucleic Acids, Columbia University Press, New York; Limbach et al., 1994 Nucleic Acids Res. 22, 2183). In an attempt to understand the biological significance, structural and thermodynamic properties, and nuclease resistance of these nucleoside modifications in nucleic acids, several, investigators have chemically synthesized nucleosides, nucleotides and phosphoramidites with various base and sugar modifications and incorporated them into oligonucleotides.
Uhlmann and Peyman, 1990, Chem. Reviews 90, 543, review the use of certain nucleoside modifications to stabilize antisense oligonucleotides.
Usman et al., International PCT Publication Nos. WO/93/15187; and WO 95/13378; describe the use of sugar, base and backbone modifications to enhance the nuclease stability of enzymatic nucleic acid molecules.
Eckstein et al., International PCT Publication No. WO 92/07065 describe the use of sugar, base and backbone modifications to enhance the nuclease stability of enzymatic nucleic acid molecules.
Grasby et al., 1994, Proc. Indian Acad. Sci., 106, 1003, review the xe2x80x9capplications of synthetic oligoribonucleotide analogues in studies of RNA structure and functionxe2x80x9d.
Eaton and Pieken, 1995, Annu. Rev. Biochem., 64, 837, review sugar, base and backbone modifications that enhance the nuclease stability of RNA molecules
Mitsunobu, 1981, Synthesis, 1, 1-28, described a process for the conversion of alcohol (ROH) to aminooxy alcohol (RONH2).
The process described by Mitsunobu (supra) has been applied in the conversion of sugars and disaccharides (Grochowski et al., 1976, 50, C15; Synthesis 1976, 682; J. Bull. Pol. Acad. Sci. Chem. Commun., 1987, 35, 255; Tronchet et al., 1982, Helv. Shim. Acta., 65, 1404; Carbohydr. Res., 1990, 204).
The process described by Mitsunobu (supra) has also been applied in the synthesis of 3xe2x80x2-Oxe2x80x94NH2 nucleosides and 5xe2x80x2-Oxe2x80x94NH2 nucleosides (Nielsen, 1995, Annu. Rev. Biomol. Struc., 24, 167; Burgess et al., 1994, J. Chem. Soc. Chem. Commun., 915; Kondo et al., 1985, Am. Chem. Soc. Symp. Ser., 16, 93; Vasseur et al., 1992, J. Am. Chem. Soc., 114, 4006; Tronchet et al., 1994, 13, 2071; Perbost et al., 1995, J. Org. Chem., 60, 5150).
The references cited above are distinct from the presently claimed invention since they do not disclose and/or contemplate the synthesis of the nucleoside analogs, such as the 2xe2x80x2-O-amino nucleosides of the instant invention.
This invention relates to a nucleoside or a nucleotide comprising a nucleic acid sugar portion, wherein the 2xe2x80x2 position of the sugar has the formula: 2xe2x80x2-Oxe2x80x94NHR1, wherein R1 is independently H, aminoacyl group, peptidyl group, biotinyl group, cholesteryl group, lipoic acid residue, retinoic acid residue, folic acid residue, ascorbic acid residue, nicotinic acid residue, 6-aminopenicillanic acid residue, 7-aminocephalosporanic acid residue, alkyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide or ester. The invention also relates to a nucleoside or a nucleotide comprising a nucleic acid sugar portion, wherein the 2xe2x80x2 position of the sugar has the formula: 2xe2x80x2-Oxe2x80x94N=R3, wherein R3 is independently pyridoxal residue, pyridoxal-5-phosphate residue, 13-cis-retinal residue, 9-cis-retinal residue, alkyl, alkenyl, alkynyl, alkylaryl, carbocyclic alkylaryl, or heterocyclic alkylaryl.
This invention relates to novel nucleoside analogs having the Formula I: 
wherein, R1 is independently H, aminoacyl group, peptidyl group, biotinyl group, cholesteryl group, lipoic acid residue, retinoic acid residue, folic acid residue, ascorbic acid residue, nicotinic acid residue, 6-aminopenicillanic acid residue, 7-aminocephalosporanic acid residue, alkyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide or ester; X is independently a nucleotide base or its analog or hydrogen; Y is independently a phosphorus-containing group; and R2 is independently a blocking group or a phosphorus-containing group.
In a preferred embodiment the invention features novel nucleoside analogs having the Formula II: 
wherein, R3 is independently pyridoxal residue, pyridoxal-5-phosphate residue, 13-cis-retinal residue, 9-cis-retinal residue, alkyl, alkenyl, alkynyl, alkylaryl, carbocyclic alkylaryl, or heterocyclic alkylaryl; X is independently a nucleotide base or its analog or hydrogen; Y is independently a phosphorus-containing group; and R2 is independently a blocking group or a phosphorus-containing group.
Specifically, an xe2x80x9calkylxe2x80x9d group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxy, cyano, alkoxy, NO2 or N(CH3)2, amino, or SH.
The term xe2x80x9calkenylxe2x80x9d group refers to unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, NO2, halogen, N(CH3)2, amino, or SH.
The term xe2x80x9calkynylxe2x80x9d refers to an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, xe2x95x90O, xe2x95x90S, NO2 or N(CH3)2, amino or SH.
An xe2x80x9carylxe2x80x9d group refers to an aromatic group which has at least one ring having a conjugated xcfx80 electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) on aryl groups are halogen, trihalomethyl, hydroxyl, SH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups.
An xe2x80x9calkylarylxe2x80x9d group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above).
xe2x80x9cCarbocyclic arylxe2x80x9d groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted.
xe2x80x9cHeterocyclic arylxe2x80x9d groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted.
An xe2x80x9camidexe2x80x9d refers to an xe2x80x94C(O)xe2x80x94NHxe2x80x94R, where R is either alkyl, aryl, alkylaryl or hydrogen.
An xe2x80x9cesterxe2x80x9d refers to an xe2x80x94C(O)xe2x80x94ORxe2x80x2, where R is either alkyl, aryl, or alkylaryl.
A xe2x80x9cblocking groupxe2x80x9d is a group which is able to be removed after polynucleotide synthesis and/or which is compatible with solid phase polynucleotide synthesis.
A xe2x80x9cphosphorus containing groupxe2x80x9d can include phosphorus in forms such as dithioates, phosphoramidites and/or as part of an oligonucleotide.
In a preferred embodiment, the invention features a process for synthesis of novel nucleoside analogs of formula I and/or Formula II.
In yet another preferred embodiment, the invention features the incorporation of novel nucleoside analogs of Formula I, Formula II or combinations thereof, into polynucleotides. These novel nucleoside analogs can be incorporated into polynucleotides enzymatically. For example by using bacteriophage T7 RNA polymerase, these novel nucleoside analogs can be incorporated into RNA at one or more positions (Milligan et al., 1989, Methods Enzymol., 180, 51). Alternatively, novel nucleoside analogs can be incorporated into polynucleotides using solid phase synthesis (Brown and Brown, 1991, in Oligonucleotides and Analogues: A Practical Approach, p. 1, ed. F. Eckstein, Oxford University Press, New York; wincott et al., 1995, Nucleic Acids Res., 23, 2677; Beaucage and Caruthers, 1996, in Bioorganic Chemistry: Nucleic Acids, p 36, ed. S. M. Hecht, Oxford University Press, New York).
The novel nucleoside analogs of Formula I, Formula II or combinations thereof, can be used for chemical synthesis of nucleotides, nucleotide-triphosphates and/or phosphoramidites as building blocks for selective incorporation into oligonucleotides. These oligonucleotides can be used as an antisense molecule, 2-5A antisense chimera, triplex forming oligonucleotides (TFO) or as an enzymatic nucleic acid molecule. The oligonucleotides can also be used as probes or primers for synthesis and/or sequencing of RNA or DNA.
Nucleosides of the instant invention can be readily converted into nucleotides, nucleotide diphosphate and nucleotide triphosphates using standard protocols (for a review see. Hutchinson, 1991, in Chemistry of Nucleosides and Nucleotides, v.2, pp 81-160, Ed. L. B. Townsend, Plenum Press, New York, USA; incorporated by reference herein).
The novel nucleoside analogs of Formula I, Formula II or combinations thereof, can also be independently or in combination used as an antiviral, anticancer or an antitumor agent. These compounds can also be independently or in combination used with other antiviral, anticancer or an antitumor agents.
By xe2x80x9cantisensexe2x80x9d it is meant a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review see Stein and Cheng, 1993 Science 261,1004).
By xe2x80x9c2-5A antisense chimeraxe2x80x9d it is meant, an antisense oligonucleotide containing a 5xe2x80x2 phosphorylated 2xe2x80x2-5xe2x80x2-linked adenylate residues. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2-5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300).
By xe2x80x9ctriplex forming oligonucleotides (TFO)xe2x80x9d it is meant an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix. Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci. USA 89, 504).
By xe2x80x9cenzymatic nucleic acidxe2x80x9d it is meant a nucleic acid molecule capable of catalyzing reactions including, but not limited to, site-specific cleavage and/or ligation of other nucleic acid molecules, cleavage of peptide and amide bonds, and trans-splicing.
The enzymatic nucleic acid is able to intramolecularly or intermolecularly cleave RNA or DNA and thereby inactivate a target RNA or DNA molecule. The enzymatic nucleic acid molecule that has complementarity in a substrate binding region to a specified gene target, also has an enzymatic activity that specifically cleaves RNA or DNA in that target. This complementarity functions to allow sufficient hybridization of the enzymatic RNA molecule to the target RNA or DNA to allow the cleavage to occur. 100% Complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. The nucleic acids may be modified at the base, sugar, and/or phosphate groups.
The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, minizyme, leadzyme oligozyme, or DNA enzyme.
By xe2x80x9ccomplementarityxe2x80x9d is meant a nucleic acid that can form hydrogen bond(s) with other RNA sequence by either traditional Watson-Crick or other non-traditional types (for example, Hoogsteen type) of base-paired interactions.
Nucleic acid molecules having an endonuclease enzymatic activity are able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence-specific manner. Such enzymatic RNA molecules can be targeted to virtually any RNA transcript, and efficient cleavage achieved in vitro (Zaug et al., 324, Nature 429 1986; Kim et al., 84 Proc. Natl. Acad. Sci. USA 8788, 1987; Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989).
Because of their sequence-specificity, trans-cleaving ribozymes show promise as therapeutic agents for human disease (Usman and McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037). Ribozymes can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the mRNA non-functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited.
Seven basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table I summarizes some of the characteristics of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
The enzymatic nature of a ribozyme is advantageous over other technologies, since the effective concentration of ribozyme necessary to effect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base-pairing. Thus, it is thought that the specificity of action of a ribozyme is greater than that of antisense oligonucleotide binding the same RNA site.
In one of the preferred embodiments of the inventions herein, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis xcex4 virus, group I intron, group II intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA. Examples of such hammerhead motifs are described by Dreyfus, supra, Rossi et al., 1992, AIDS Research and Human Retroviruses 8, 183; of hairpin motifs by Hampel et al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein et al., 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene, 82, 43, and Hampel et al., 1990 Nucleic Acids Res. 18, 299; of the hepatitis xcex4 virus motif is described by Perrofta and Been, 1992 Biochemistry 31, 16; of the RNaseP motif by Guerrier-Takada et al., 1983 Cell 35, 849; Forster and Altman, 1990, Science 249, 783; Li and Altman, 1996, Nucleic Acids Res. 24, 835; Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993 Biochemistry 32, 2795-2799; Guo and Collins, 1995, EMBO. J. 14, 363); Group II introns are described by Griffin et al., 1995, Chem. Biol. 2, 761; Michels and Pyle, 1995, Biochemistry 34, 2965; Pyle et al., International PCT Publication No. WO 96/22689; and of the Group I intron by Cech et al., U.S. Pat. No. 4,987,071. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.
Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small nucleic acid motifs (e.g., antisense oligonucleotides, hammerhead or the hairpin ribozymes) are used for exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of the mRNA structure.
The novel nucleoside analogs of the instant invention and/or the polynucleotides comprising these analogs are added directly to a cell, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells. The nucleosides, nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stent, with or without their incorporation in biopolymers.
By xe2x80x9cenzymatic portionxe2x80x9d is meant that part of the ribozyme essential for cleavage of an RNA substrate.
By xe2x80x9csubstrate binding armxe2x80x9d is meant that portion of a ribozyme which is complementary to (ie., able to base-pair with) a portion of its substrate. Generally, such complementarity is 100%, but can be less if desired. For example, as few as 10 bases out of 14 may be base-paired. Such arms are shown generally in FIGS. 1-3 as discussed below. That is, these arms contain sequences within a ribozyme which are intended to bring ribozyme and target RNA together through complementary base-pairing interactions; e.g., ribozyme sequences within stems I and III of a standard hammerhead ribozyme make up the substrate-binding domain (see FIG. 1).
By xe2x80x9coligonucleotidexe2x80x9d or xe2x80x9cpolynucleotidexe2x80x9d as used herein, is meant a molecule comprising two or more nucleotides.
By xe2x80x9cunmodified nucleosidexe2x80x9d is meant one of the bases adenine, cytosine, guanine, uracil joined to the 1xe2x80x2 carbon of xcex2-D-ribo-furanose.
By xe2x80x9cmodified nucleosidexe2x80x9d is meant any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate.
Oligonucleotide conjugates with different types of biologically interesting macromolecules or reporter groups are useful as experimental tools. In this invention, the existence of a 2xe2x80x2-O-amino functionality facilitates formation of such conjugates. A majority of the methods known in the art for the chemical synthesis of such conjugates are based either on post-synthetic attachment of the molecule of interest to the 3xe2x80x2- and/or 5xe2x80x2-end of oligonucleotides using an appropriate spacer, or incorporation of the sugar, base and/or backbone-modified, monomeric nucleoside units into oligonucleotides during chemical synthesis. However, these methods have several disadvantages such as low yields and tedious synthesis schemes. To avoid these problems it is useful to use unique functional groups both in the oligonucleotides and in the molecule to be attached. These functional groups should be able to react quantitatively, under mild conditions and preferably in water solution. The main idea here is to design a nucleoside monomeric unit (phosphoramidite) bearing a unique functional group, which can be further used as a tether for conjugating any molecule of interest.
Formation of oximes (interaction of aldehydes or ketones with hydroxylamines) or oxyamides (interaction of carboxylic acids with hydroxylamines) are the organic reactions of choice which will meet the above requirements (Sandler, S. R.; Karo, W Organic Functional Group Preparation vol. III, ed. Wasserman H. H., Academic Press, Inc., 1989, pp 378-523).
In a preferred embodiment, an oligonucleotide would bear one or more hydroxylamino functionalities attached directly to the monomeric unit or through the use of an appropriate spacer (e.g., an alkyl moiety or any moiety which does not significantly interfere with the action of the nucleotide or the hydroxylamine functionality). Since oligonucleotides have neither aldehyde nor hydroxylamino groups, the formation of an oxime would occur selectively using oligo as a polymeric template. This approach would facilitate the attachment of practically any molecule of interest (peptides, polyamines, coenzymes, oligosaccharides, lipids, etc.) directly to the oligonucleotide using either aldehyde or carboxylic function in the molecule of interest. 
The oximation reaction proceeds in water
Quantitative yields
Hydrolytic stability in a wide pH range (5-8)
The amphoteric nature of oximes allows them to act either as weak acids or weak bases.
Oximes exhibit a great tendency to complex with metal ions
In yet another preferred embodiment, the aminooxy xe2x80x9ctetherxe2x80x9d in oligonucleotides, such as a ribozyme, is reacted with different compounds bearing carboxylic groups (e.g. aminoacids, peptides, xe2x80x9ccapxe2x80x9d structures, etc.) resulting in the formation of oxyamides as shown below. 
In a preferred embodiment the invention features a process for the synthesis of a 2xe2x80x2-O-amino nucleoside, such as 2xe2x80x2-O-amino adenosine, 2xe2x80x2-O-amino guanosine, 2xe2x80x2-O-amino cytidine, 2xe2x80x2-O-amino uridine and others, comprising the steps of: a) contacting a 3xe2x80x2 and 5xe2x80x2-protected arabino nucleoside with a sulfonylating reagent, such as tri-fluoromethane sulfonic anhydride, trifluoromethane sulfonic chloride and others, under conditions suitable for the formation of 3xe2x80x2 and 5xe2x80x2-protected 2xe2x80x2-arabino sulfonyl nucleoside; b) displacement of the sulfonyl group from the 2xe2x80x2-arabino sulfonyl nucleoside with N-hydroxyphthalimide in the presence of a strong organic base, such as 1,8-diazabicyclo(5.4.0)undec-7-ene and the like, under conditions suitable for the formation of 3xe2x80x2 and 5xe2x80x2-protected 2xe2x80x2-Oxe2x80x94N-phthaloyl ribonucleoside; c) deprotection of the N-phthaloyl ribonucleoside with a fluoride containing reagent, such as tetrabutylammonium fluoride, triethylamine trihydrofluoride and the like, under conditions suitable for the formation of 2xe2x80x2-Oxe2x80x94N-phthaloyl ribonucleoside; and d) contacting the 2xe2x80x2-Oxe2x80x94N-phthaloyl ribonucleoside with a reagent selected from a group consisting of alkylamine (such as methylamine, ethylamine, butylamine and the like), hydrazine, N-phenyl hydrazine and Nalkylhydrazine (such as N-methylhydrazine and the like), under conditions suitable for the formation of said 2xe2x80x2-O-amino nucleoside.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.